Abstract
Methods that facilitate the accurate counting of specific neural cell types would be of substantial value in evaluating the efficacy of treatments applied to spinal cord injury. This report describes reliable procedures for identification of neurons, oligodendrocytes, astrocytes, endothelial cells and inflammatory cells (neutrophils and activated macrophage/microglial cells) in paraformaldehyde-fixed, paraffin-embedded injured adult rat spinal cord. Antigen retrieval techniques (enzymatic and thermal) were used to improve antibody access to masked epitopes. To decrease background immunofluorescence and autofluorescence of hemoglobin, the tissue sections were pretreated with 0.1% sodium borohydride in PBS (30 min), followed by 1-5 min incubation in 0.5% Sudan black in 70% ethanol. Commercially available techniques to amplify the primary signal such as tyramide signal amplification (TSA) and avidin/biotin/peroxidase/ DAB/nickel/cobalt amplification (ABP/DABA) were also tested. Hoechst 33342 nuclear staining was used to indicate cell location, number, and integrity, thereby avoiding misidentification of cells. The best antibodies were: anti-NeuN antibody for neurons, anti-S100 for astrocytes, and anti-S100 and APC-7 antibodies in combination for oligodendrocytes, anti-laminin (LN) for endothelial cells, and ED1 antibody for activated macrophages and microglia. Amplification of the primary signal with TSA or ABP/DABA was also found to be beneficial.
| Original language | English |
|---|---|
| Pages (from-to) | 1-11 |
| Number of pages | 11 |
| Journal | Journal of Neuroscience Methods |
| Volume | 139 |
| Issue number | 1 |
| DOIs | |
| State | Published - Oct 15 2004 |
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Keywords
- Antigen retrieval
- Autofluorescence
- Hoechst staining
- Laminin
- Signal amplification
ASJC Scopus subject areas
- Neuroscience(all)
Cite this
Improved immunocytochemical identification of neural, endothelial, and inflammatory cell types in paraffin-embedded injured adult rat spinal cord. / Casella, Gizelda T B; Bunge, Mary B; Wood, Patrick.
In: Journal of Neuroscience Methods, Vol. 139, No. 1, 15.10.2004, p. 1-11.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Improved immunocytochemical identification of neural, endothelial, and inflammatory cell types in paraffin-embedded injured adult rat spinal cord
AU - Casella, Gizelda T B
AU - Bunge, Mary B
AU - Wood, Patrick
PY - 2004/10/15
Y1 - 2004/10/15
N2 - Methods that facilitate the accurate counting of specific neural cell types would be of substantial value in evaluating the efficacy of treatments applied to spinal cord injury. This report describes reliable procedures for identification of neurons, oligodendrocytes, astrocytes, endothelial cells and inflammatory cells (neutrophils and activated macrophage/microglial cells) in paraformaldehyde-fixed, paraffin-embedded injured adult rat spinal cord. Antigen retrieval techniques (enzymatic and thermal) were used to improve antibody access to masked epitopes. To decrease background immunofluorescence and autofluorescence of hemoglobin, the tissue sections were pretreated with 0.1% sodium borohydride in PBS (30 min), followed by 1-5 min incubation in 0.5% Sudan black in 70% ethanol. Commercially available techniques to amplify the primary signal such as tyramide signal amplification (TSA) and avidin/biotin/peroxidase/ DAB/nickel/cobalt amplification (ABP/DABA) were also tested. Hoechst 33342 nuclear staining was used to indicate cell location, number, and integrity, thereby avoiding misidentification of cells. The best antibodies were: anti-NeuN antibody for neurons, anti-S100 for astrocytes, and anti-S100 and APC-7 antibodies in combination for oligodendrocytes, anti-laminin (LN) for endothelial cells, and ED1 antibody for activated macrophages and microglia. Amplification of the primary signal with TSA or ABP/DABA was also found to be beneficial.
AB - Methods that facilitate the accurate counting of specific neural cell types would be of substantial value in evaluating the efficacy of treatments applied to spinal cord injury. This report describes reliable procedures for identification of neurons, oligodendrocytes, astrocytes, endothelial cells and inflammatory cells (neutrophils and activated macrophage/microglial cells) in paraformaldehyde-fixed, paraffin-embedded injured adult rat spinal cord. Antigen retrieval techniques (enzymatic and thermal) were used to improve antibody access to masked epitopes. To decrease background immunofluorescence and autofluorescence of hemoglobin, the tissue sections were pretreated with 0.1% sodium borohydride in PBS (30 min), followed by 1-5 min incubation in 0.5% Sudan black in 70% ethanol. Commercially available techniques to amplify the primary signal such as tyramide signal amplification (TSA) and avidin/biotin/peroxidase/ DAB/nickel/cobalt amplification (ABP/DABA) were also tested. Hoechst 33342 nuclear staining was used to indicate cell location, number, and integrity, thereby avoiding misidentification of cells. The best antibodies were: anti-NeuN antibody for neurons, anti-S100 for astrocytes, and anti-S100 and APC-7 antibodies in combination for oligodendrocytes, anti-laminin (LN) for endothelial cells, and ED1 antibody for activated macrophages and microglia. Amplification of the primary signal with TSA or ABP/DABA was also found to be beneficial.
KW - Antigen retrieval
KW - Autofluorescence
KW - Hoechst staining
KW - Laminin
KW - Signal amplification
UR - http://www.scopus.com/inward/record.url?scp=4444297827&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=4444297827&partnerID=8YFLogxK
U2 - 10.1016/j.jneumeth.2004.04.008
DO - 10.1016/j.jneumeth.2004.04.008
M3 - Article
VL - 139
SP - 1
EP - 11
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
SN - 0165-0270
IS - 1
ER -